A broad goal of neuroscience is to understand decision making in animal behavior, from sensory input, to neuronal response and computation, to the resulting behavioral output. Obtaining this sort of knowledge in humans is extremely complicated, but understanding the process for a well-defined stimulus like temperature, in simpler animals like Drosophila, is a tractable and highly rewarding undertaking. Particularly in their larval form, Drosophila behavior as they navigate their environment is highly quantifiable; they are also well-suited for in vivo optical imaging, and we can observe activity at the single-neuron level. We propose a detailed investigation into the neurobiology of larval temperature response, both at the level of observing navigation strategies across larval developmental stages, and at the neuronal level as we identify neurons that participate in temperature response, examine their sensitivity, and determine what changes over development. The behavioral component can be achieved with novel temperature control apparatuses and data acquisition methods capable of precisely quantifying navigation strategy; the imaging component will utilize a modified 3D spinning-disk confocal microscope, its high contrast allowing single-neuron resolution and its sensitivity allowing detection of calcium activity as neurons respond to temperature variation. Technical advances will enable this work to resolve several disputed questions in the field and increase our broader understanding of animal behavior and decision-making. Using this quantitative behavioral analysis and optical neurophysiology, we will characterize the precise contribution of molecules that have been implicated in thermotaxis to particular components of thermotaxis, from perception to navigational decision-making. It is known that transient receptor potential (TRP) channels are involved in heat and cold avoidance, and TRP TRP channels have a special relevance to human health, as such channels are also found in mammals, and mutations in some TRP channels underlie diseases. PUBLIC HEALTH RELEVANCE: To better understand the connection between stimuli and neuronal responses in more complicated animals like humans, it is helpful to start with computational circuits in simpler animals. In particular here, precise and quantitative insight int sensory encoding of temperature in the Drosophila larva will expand our knowledge of behavioral and neural circuit function and development. This will be researched using sophisticated techniques in quantitative behavioral analysis and neuronal imaging. A comprehensive understanding of these simple circuits and the rules that govern them will help us to determine the mechanisms of more complex circuits that regulate more elusive, sophisticated behaviors, such as mood and attention, and how these circuits go awry in human disease.